Christopher J. Phiel, Ph.D. The presence of methylated adenosines (referred to as m6A) in mRNA had been discovered 40 years ago, but the functional significance of m6A remained elusive. Recent breakthroughs have identified the molecular mediators that establish and remove the m6A mark. In addition, the presence of m6A on mRNA was found to be a signal to degrade mRNA. When m6A levels are reduced in embryonic stem cells (ESCs) by manipulation of the factors involved in the m6A process, the ESCs are unable to properly differentiate, and remain in a pluripotent state. Interestingly, the inability to differentiate is the same phenotype found in ESCs in which the genes Gsk-3? and Gsk-3? are genetically deleted. Gsk-3? and Gsk-3? are key kinases that phosphorylate a variety of proteins. Based on the similarity in phenotypes between Mettl3 and Gsk-3?/Gsk-3? double knockout (DKO) ESCs, we hypothesize that the process of mRNA m6A modification is regulated by Gsk-3?/Gsk-3? activity. Specifically, we hypothesize that Gsk-3?/Gsk-3? normally phosphorylates the m6A demethylase FTO, targeting the protein for ubiquitin-mediated degradation; in the absence of Gsk-3?/Gsk-3?, FTO levels are raised resulting in reduced m6A levels, leading to a persistent pluripotent state in ESCs. The following specific aims are designed to test this hypothesis: Specific Aim 1. Determine the precise mechanism by which Gsk-3 activity regulates FTO in ESCs. Specific Aim 2. Identify the specific mRNAs that have reduced m6A modifications in Gsk-3 DKO ESCs. Specific Aim 3. Investigate which Gsk-3-dependent signaling pathways affect m6A levels. The discovery that the m6A modification of mRNA is controlled by a multi-faceted regulator of signal transduction represents the first mechanistic insight into how this fundamental process is regulated, and would have profound consequences for understanding how different Gsk-3-dependent signaling pathways regulate the balance between pluripotency and differentiation in embryonic stem cells.